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Mass Spectrometric Methods for Analysis of Sterols and Steryl Esters in Biological Samples

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Mass Spectrometric Methods for Analysis of Sterols and Steryl Esters in Biological Samples Mass Spectrometric Methods for Analysis of Sterols and Steryl Esters in Biological Samples by © Iyad Ali Hailat A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry Faculty of Science Memorial University of Newfoundland May 2014 St. John’s Newfoundland i Abstract The composition of free sterols in cultivated blue mussels obtained commercially and from laboratory feeding experiments has been investigated. Total lipids were extracted from mussels using a modified Folch procedure followed by isolation of free sterols using column chromatography. Sterols were identified using GC/MS and quantified using (GC/MS-SIM) and GC/FID. The most abundant free sterols in commercially cultivated mussels were cholesterol, desmosterol, and brassicasterol. Mussels were fed on algae or fish waste for six months. The concentrations of sterols in fish waste-fed mussels were compared with those in algae-fed mussels. However, fish waste-fed mussels had significantly higher cholesterol concentrations compared to algae- fed mussels. Mussels fed algae had significantly higher campesterol compared to fish waste-fed mussels. Free sterols are neutral molecules that are difficult to ionize by MALDI or ESI. Therefore, in order to increase their ionization efficiency, sterols were converted into their corresponding picolinyl esters, N-methylpyridyl ethers and sulphated esters. Using MALDI-MS, sterol picolinyl esters were identified as [M+Na]+, the signal being significantly enhanced after the addition of sodium acetate. Sterol N-methylpyridyl ethers were detected as [M]+ while sulphated sterols were detected as [M-H]-. The ester bonds of picolinyl and sulphated esters cleaved during MALDI-CID MS/MS resulting in diagnostic fragments at m/z 146 and 97, respectively. ii Picolinyl esters of sterols were also analyzed using ESI/APCI-QIT MSn. The Picolinyl esters were detected as [M+H]+ in ESI and APCI sources. The ester bonds of picolinyl esters cleaved during CID MS2, resulting in diagnostic fragments corresponding + 3 + to steryl cation moieties [M+H-C6H5NO2] . The CID MS of [M+H] → [M+H- + C6H5NO2] of picolinyl esters was found to be useful for structural elucidation and for distinguishing among steryl isomers. Steryl esters were identified in margarine and corn using ESI-QIT MSn. Sterols and other lipids extracted from samples using hexane were subjected to solid phase extraction. The steryl ester fraction was eluted with hexane: diethyl ether (98:2, v/v). Fatty acid steryl + esters were detected as [M+NH4] using ammonium acetate as dopant. Steryl esters, including molecular isomers, were identified using ESI-QIT MS2. ESI-QIT MS3 was carried out on the intact steryl fragmentation cation. The resulting CID spectra of the steryl cation were found to be unique and similar to those from APCI-QIT MS2 CID of free sterol standards. iii Acknowledgments First of all, I am greatly thankful to my supervisory committee members, Dr. Christopher Parrish and Dr. Peter Pickup for their support and advice. I would like to acknowledge the Chemistry Department, the Ocean Sciences Center (OSC), and the Center for Chemical Analysis Research and Training (C-CART) for training on instruments. I would like also to thank Ms. Linda Winsor from C-CART, and Ms. Jeannette Wells from OSC for training and helping me in analyzing of samples. I would like to acknowledge Memorial University of Newfoundland and National Sciences and Engineering Research Council of Canada (NSERC) for funding. I would like to thank the members of analytical chemistry group for sharing their ideas and for lively discussions. I would like to express my deep appreciation and thanks to my mother, brothers and sisters for their encouragement and support. Finally, I would like to express my deep thanks to my supervisors, Dr. Robert Helleur and Christopher Parrish, for their guidance, support, encouragement and patience during this work. iv Dedication To the soul of my father To my mother, brothers and sisters v Table of Contents Title……………………………………………………………………………………….. i Abstract…………………………………………………………………………………... ii Acknowledgements……………………………………………………………………… iv Dedication………………………………………………………………………………....v Table of contents………………………………………………………………………….vi List of tables………………………………………………………………………………xi List of figures…………………………………………………………………………….xii List of Abbreviations……………………………………………………………………..xv Chapter One: Introduction and Overview.…………………………………………......1 1.1 Lipids and their chemical structures…………………………………………………...2 1.2 Chemistry of sterols……………………………………………………………………4 1.3 Abbreviation system in sterols……………………………………………………….10 1.4 Conjugated sterols……………………………………………………………………13 1.5 The importance of sterols…………………………………………………………….15 1.6 Sterols in mariculture………………………………………………………………...15 1.6.1 The origin of sterols in molluscs and bivalves……………………………...15 1.6.2 Sterol composition of molluscs……………………………………………..17 1.6.3 Sterols and their biosynthesis in mussels…………………………………...17 1.6.4 Sterols in microalgae………………………………………………………..18 1.7 A brief introduction to mass spectrometry…………………………………………...19 1.7.1 Types of ionization techniques…………………………………………......20 1.7.2 Electrospray ionization mass spectrometry………………………………...21 vi 1.7.3 Atmospheric pressure chemical ionization…………………………………23 1.7.4. Matrix-assisted laser desorption/ionization (MALDI)…………………….24 1.7.4.1 Basic concepts…………………………………………………….24 1.7.4.2 The role of matrix………………………………………………....26 1.7.4.3 The addition of the salts as dopants in MALDI-TOFMS………...27 1.7.4.4 Time-of-flight (TOF) mass analyzer……………………………...27 1.8 Quadrupole ion-trap mass spectrometry……………………………………………..29 1.9 Tandem mass spectrometry…………………………………………………………..33 1.9.1 Types of tandem scans……………………………………………………....34 1.9.2 Collision-induced dissociation……………………………………………....35 1.9.3 Tandem mass spectrometry using a quadrupole ion trap MS……………….35 1.10 Analysis of free sterols and steryl esters……………………………………………36 1.10.1 Preparative chromatography………………………………………………..37 1.10.1.1 Coulmn chromatography (CC)…………………………………..37 1.10.1.2 Thin-layer chromatography (TLC)………………………………37 1.10.1.3 Solid phase extraction (SPE)………………………………….....38 1.10.2 Techniques for analysis of free sterols and steryl esters……………………39 1.10.2.1 Analysis of sterols using ESI-MS and APCI-MS…………….....40 1.10.2.2 Analysis of sterols by derivatization MALDI-TOF……………..42 1.10.2.3 Analysis of steryl esters…………………………………………42 1.11 Thesis objectives…………………………………………………………………...44 1.12 Co-authorship statement…………………………………………………………..45 1.13 References…………………………………………………………………………46 Chapter Two- Lipid and sterol compositions of blue mussels fed algae and culture finfish effluent diets…………………………………………………………………….55 2.1 Introduction………………………………………………………………………....56 vii 2.2 Materials and methods……………………………………………………………….59 2.2.1 Feeding experiments………………………………………………………59 2.2.2 Chemical reagents…………………………………………………………59 2.2.3 Lipid extraction……………………………………………………………60 2.2.4 Lipid classes……………………………………………………………….60 2.2.5 Free sterols………………………………………………………………...61 2.2.6 Gas chromatographic separation and mass spectrometric detection condition of sterols………………………………………………………………………….62 2.2.7 Statistical analysis………………………………………………………….63 2.3 Results and Discussion……………………………………………………………....63 2.3.1 Lipid content……………………………………………………………..63 2.3.2 Free sterol composition of cultivated mussels……………………………64 2.3.3 GC-MS selected-ion monitoring (GC-MS-SIM)…………………………66 2.3.4. The concentrations of sterols in diets used in this study………………...66 2.3.5 Sterols in unfed mussels and those fed on algae or culture finfish effluent (fish waste)……………………………………………………………………...68 2.4 Conclusion……………………………………………………………………………74 2.5 References……………………………………………………………………………75 Chapter Three - Direct analysis of derivatized sterols using mass spectrometry and tandem mass spectrometry……………………………………………………………..78 3.1 Introduction……………………………………………………………………….....79 3.2 Experimental………………………………………………………………………....83 3.2.1 Chemicals and reagents………………………………………………….83 3.2.2 Derivatization of sterols……………………………………………….....83 3.2.2.1 Picolinyl esters………………………………………………..83 3.2.2.2 N-Methylpyridyl ethers…………………………………….....84 3.2.2.3 Sulphated esters……………………………………………....84 3.2.3 Sample preparation for MALDI-TOF analysis…………………………..85 viii 3.2.3.1 Free sterols…………………………………………………....85 3.2.3.2 Sterol picolinyl esters…………………………………………86 3.2.3.3 Sterol N-methylpyridyl ethers ……………………………......86 3.2.3.4 Sterol sulphates……………………………………………….86 3.2.4 Quantification of derivatized sterols……………………………………..86 3.2.5 MALDI-TOF Instrumentation…………………………………………...87 3.2.6 ESI-QIT MS and APCI-QIT MS of picolinyl ester of sterols…………...87 3.3 Results and Discussions……………………………………………………………...88 3.3.1 Direct analysis of sterols by derivatization MALDI-TOF mass spectrometry and tandem mass spectrometry……………………………………………………...88 3.3.1.1 MALDI-TOF analysis………………………………………....88 3.3.1.1.1 Free sterols…………………………………………88 3.3.1.1.2 Picolinyl esters……………………………………..89 3.3.1.1.3 N-Methylpyridyl ethers…………………………….89 3.3.1.1.4 Sterol sulphates…………………………………….92 3.3.1.2 MALDI-TOF/TOF analysis…………………………………..94 3.3.1.3 Reproducibility and sensitivity……………………………….96 3.3.1.4 Identification of sterols in blue mussels………………………97 3.3.1.5 Calibration for derivatives and sterol quantification in mussels..101 3.3.2 Analysis of sterols as their picolinyl esters using both flow-injection ESI- QIT MSn and APCI-QIT MSn………………………………………….103
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